Adaptive discriminant function analysis and reranking of MS/MS database search results for improved peptide identification in shotgun proteomics

Robust statistical validation of peptide identifications obtained by tandem mass spectrometry and sequence database searching is an important task in shotgun proteomics. PeptideProphet is a commonly used computational tool that computes confidence measures for peptide identifications. In this paper, we investigate several limitations of the PeptideProphet modeling approach, including the use of fixed coefficients in computing the discriminant search score and selection of the top scoring peptide assignment per spectrum only. To address these limitations, we describe an adaptive method in which a new discriminant function is learned from the data in an iterative fashion. We extend the modeling framework to go beyond the top scoring peptide assignment per spectrum. We also investigate the effect of clustering the spectra according to their spectrum quality score followed by cluster-specific mixture modeling. The analysis is carried out using data acquired from a mixture of purified proteins on four different types of mass spectrometers, as well as using a complex human serum data set. A special emphasis is placed on the analysis of data generated on high mass accuracy instruments.     

Compositional protein analysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS and probabilistic peptide scoring

A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.     

A new algorithm for the evaluation of shotgun peptide sequencing in proteomics: support vector machine classification of peptide MS/MS spectra and SEQUEST scores

Shotgun tandem mass spectrometry-based peptide sequencing using programs such as SEQUEST allows high-throughput identification of peptides, which in turn allows the identification of corresponding proteins. We have applied a machine learning algorithm, called the support vector machine, to discriminate between correctly and incorrectly identified peptides using SEQUEST output. Each peptide was characterized by SEQUEST-calculated features such as delta Cn and Xcorr, measurements such as precursor ion current and mass, and additional calculated parameters such as the fraction of matched MS/MS peaks. The trained SVM classifier performed significantly better than previous cutoff-based methods at separating positive from negative peptides. Positive and negative peptides were more readily distinguished in training set data acquired on a QTOF, compared to an ion trap mass spectrometer. The use of 13 features, including four new parameters, significantly improved the separation between positive and negative peptides. Use of the support vector machine and these additional parameters resulted in a more accurate interpretation of peptide MS/MS spectra and is an important step toward automated interpretation of peptide tandem mass spectrometry data in proteomics.     

BuildSummary: using a group-based approach to improve the sensitivity of peptide/protein identification in shotgun proteomics

The target-decoy database search strategy is widely accepted as a standard method for estimating the false discovery rate (FDR) of peptide identification, based on which peptide-spectrum matches (PSMs) from the target database are filtered. To improve the sensitivity of protein identification given a fixed accuracy (frequently defined by a protein FDR threshold), a postprocessing procedure is often used that integrates results from different peptide search engines that had assayed the same data set. In this work, we show that PSMs that are grouped by the precursor charge, the number of missed internal cleavage sites, the modification state, and the numbers of protease termini and that the proteins grouped by their unique peptide count should be filtered separately according to the given FDR. We also develop an iterative procedure to filter the PSMs and proteins simultaneously, according to the given FDR. Finally, we present a general framework to integrate the results from different peptide search engines using the same FDR threshold. Our method was tested with several shotgun proteomics data sets that were acquired by multiple LC/MS instruments from two different biological samples. The results showed a satisfactory performance. We implemented the method in a user-friendly software package called BuildSummary, which can be downloaded for free from http://www.proteomics.ac.cn/software/proteomicstools/index.htm as part of the software suite ProteomicsTools.     

PepDistiller: A quality control tool to improve the sensitivity and accuracy of peptide identifications in shotgun proteomics

In this study, we presented a quality control tool named PepDistiller to facilitate the validation of MASCOT search results. By including the number of tryptic termini, and integrating a refined false discovery rate (FDR) calculation method, we demonstrated the improved sensitivity of peptide identifications obtained from semitryptic search results. Based on the analysis of a complex data set, approximately 7% more peptide identifications were obtained using PepDistiller than using MASCOT Percolator. Moreover, the refined method generated lower FDR estimations than the percentage of incorrect target (PIT) fixed method applied in Percolator. Using a standard data set, we further demonstrated the increased accuracy of the refined FDR estimations relative to the PIT-fixed FDR estimations. PepDistiller is fast and convenient to use, and is freely available for academic access. The software can be downloaded from http://www.bprc.ac.cn/pepdistiller.     

Indoleamine 2,3-Dioxygenase Controls Fungal Loads and Immunity in Paracoccidioidomicosis but is More Important to Susceptible than Resistant Hosts

Background

Paracoccidioidomycosis, a primary fungal infection restricted to Latin America, is acquired by inhalation of fungal particles. The immunoregulatory mechanisms that control the severe and mild forms of paracoccidioidomycosis are still unclear. Indoleamine 2,3-dioxygenase (IDO), an IFN-γ induced enzyme that catalyzes tryptophan metabolism, can control host-pathogen interaction by inhibiting pathogen growth, T cell immunity and tissue inflammation.

Methodology/Principal Findings

In this study, we investigated the role of IDO in pulmonary paracoccidioidomycosis of susceptible and resistant mice. IDO was blocked by 1-methyl-dl-tryptophan (1MT), and fungal infection studied in vitro and in vivo. Paracoccidioides brasiliensis infection was more severe in 1MT treated than untreated macrophages of resistant and susceptible mice, concurrently with decreased production of kynurenines and IDO mRNA. Similar results were observed in the pulmonary infection. Independent of the host genetic pattern, IDO inhibition reduced fungal clearance but enhanced T cell immunity. The early IDO inhibition resulted in increased differentiation of dendritic and Th17 cells, accompanied by reduced responses of Th1 and Treg cells. Despite these equivalent biological effects, only in susceptible mice the temporary IDO blockade caused sustained fungal growth, increased tissue pathology and mortality rates. In contrast, resistant mice were able to recover the transitory IDO blockade by the late control of fungal burdens without enhanced tissue pathology.

Conclusions/Significance

Our studies demonstrate for the first time that in pulmonary paracoccidioidomycosis, IDO is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity and inflammation, with prominent importance to susceptible hosts. In fact, only in the susceptible background IDO inhibition resulted in uncontrolled tissue pathology and mortality rates. Our findings open new perspectives to understand the immunopathology of paracoccidioidomycosis, and suggest that an insufficient IDO activity could be associated with the severe cases of human PCM characterized by inefficient fungal clearance and excessive inflammation.     

A LC-MS/MS methodology to determine furaltadone residues in the macroalgae Ulva lactuca

Presently, the rise of new contaminants in the environment has widened the scope of pharmaceutical analyses as to face the demanding new challenges. An increasing tendency for the interconnection and overlap of research fields, such as ecology and biochemistry, is intensifying the demand for new methodologies to be applied to the survey of drugs in unconventional matrices. Integrated in this group are macrophytes, such as the green macroalgae Ulva lactuca, which are under study as to ascertain their ability as indicators of contamination for many substances. Nonetheless, methodologies for extraction and determination of drugs in such matrices are scarce and new studies on the subject are pressing. A new methodology for the determination of the antibiotic furaltadone in U. lactuca by liquid chromatography-tandem mass spectrometric (LC-MS/MS) procedure was developed, optimized and validated following the guidelines of the EC Decision 2002/657. The calibration curves showed linearity above 0.99 (R(2)). The relative standard deviations obtained for repeatability, expressed as CV, were between 15.3 and 20.5 and for reproducibility 25.3 and 28.2 whereas accuracy was in the interval of 88.9-95.5 (%). The limit of decision (CCα) and the detection capability (CCβ) were respectively 5.57 μg kg(-1) and 10.97 μg kg(-1). The method was successfully applied to experimental samples.     

A novel alignment method and multiple filters for exclusion of unqualified peptides to enhance label-free quantification using peptide intensity in LC-MS/MS

Though many software packages have been developed to perform label-free quantification of proteins in complex biological samples using peptide intensities generated by LC-MS/MS, two critical issues are generally ignored in this field: (i) peptides have multiple elution patterns across runs in an experiment, and (ii) many peptides cannot be used for protein quantification. To address these two key issues, we have developed a novel alignment method to enable accurate peptide peak retention time determination and multiple filters to eliminate unqualified peptides for protein quantification. Repeatability and linearity have been tested using six very different samples, i.e., standard peptides, kidney tissue lysates, HT29-MTX cell lysates, depleted human serum, human serum albumin-bound proteins, and standard proteins spiked in kidney tissue lysates. At least 90.8% of the proteins (up to 1,390) had CVs ≤ 30% across 10 technical replicates, and at least 93.6% (up to 2,013) had R(2) ≥ 0.9500 across 7 concentrations. Identical amounts of standard protein spiked in complex biological samples achieved a CV of 8.6% across eight injections of two groups. Further assessment was made by comparing mass spectrometric results to immunodetection, and consistent results were obtained. The new approach has novel and specific features enabling accurate label-free quantification.     

In vitro degradation of the antimicrobial human peptide HEM-gamma 130-146 in plasma analyzed by a validated quantitative LC-MS/MS procedure

In stability studies during preclinical drug development, the human antimicrobial peptide hHEM-gamma 130-146 shows progressive N-terminal degradation in plasma. To determine this effect, we developed and validated a selective and quantitative muHPLC-MS/MS procedure for this compound. Following deproteinization by precipitation, reversed-phase separation is performed with a time-saving two-column design online coupled to an ion trap mass spectrometer for electrospray ionization MS detection. Using a linear calibration curve obtained with synthetic external standards ranging nearly two orders of magnitude, we achieved good precision (repeatability and reproducibility: 5-15%), accuracy (-3 to 15%), and ruggedness with a lower limit of quantification at 0.29 microg/ml plasma (0.15 microM). Because of good linearity (r2>0.999), the recovery (84+/-3%) and ion suppression (86+/-4% remaining intensity) were calculated from specifically prepared calibration curves. The developed procedure was applied to human and animal plasma samples. Incubations in the presence and absence of proteinase inhibitors revealed at least an aminopeptidase M activity for the initial N-terminal truncation of tryptophan (W130) and a putative glutaminyl-peptide cyclotransferase activity for the resulting intermediate starting with the bared glutamine residue (Q131). The calculated periods of half-change demonstrated exceeding interspecies variations, whereas the intraspecies variations were only between 20 and 30%. The current procedure is valuable as a generic method for pharmaceutical purposes, and data give important information for further development toward a potential natural drug candidate.     

Antibody reactive to a hepatitis C virus (HCV)-derived peptide capable of inducing HLA-A2 restricted cytotoxic T lymphocytes is detectable in a majority of HCV-infected individuals without HLA-A2 restriction

Hepatitis C virus (HCV) is a single-strand RNA virus. Approximately 170 million people around the world are persistently infected and are at risk of liver cirrhosis or cancer. There is an urgent need to develop both therapeutic and diagnostic modalities of HCV. One approach to achieve these goals would be to determine highly immunodominant HCV peptides which are recognized by both cellular and humoral immunities. This study reports one such peptide, HCV-core protein at positions 35-44, having HLA-A2 binding motifs. IgG specific to this CTL-epitope peptide is consistently detectable in a majority of the patients with HCV infection regardless of the different HLA types, different disease conditions, and different HCV-genotypes tested. The sequence LPRR at positions 37-40 is considered to be the fine epitope recognized by the IgG. These results may provide new insights for the development of both therapeutic and diagnostic modalities of HCV at lower costs.     

More than one glycan is needed for ER glucosidase II to allow entry of glycoproteins into the calnexin/calreticulin cycle

Nascent and newly synthesized glycoproteins enter the calnexin (Cnx)/calreticulin (Crt) cycle when two out of three glucoses in the core N-linked glycans have been trimmed sequentially by endoplasmic reticulum (ER) glucosidases I (GI) and II (GII). By analyzing arrested glycopeptides in microsomes, we found that GI removed the outermost glucose immediately after glycan addition. However, although GII associated with singly glycosylated nascent chains, trimming of the second glucose only occurred efficiently when a second glycan was present in the chain. Consistent with a requirement for multiple glycans to activate GII, pancreatic RNase in live cells needed more than one glycan to enter the Cnx/Crt cycle. Thus, whereas GI trimming occurs as an automatic extension of glycosylation, trimming by GII is a regulated process. By adjusting the number and location of glycans, glycoproteins can instruct the cell to engage them in an individually determined folding and quality control pathway.     

Comparative study of the LC-MS/MS and UPLC-MS/MS for the multi-residue analysis of quinolones, penicillins and cephalosporins in cow milk, and validation according to the regulation 2002/657/EC

The aim of this study was to develop and validate an analytical method to simultaneously determine European Union-regulated β-lactams (penicillins and cephalosporins) and quinolones in cow milk. The procedure involves a new solid phase extraction (SPE) to clean-up and pre-concentrate the three series of antibiotics before analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). LC-MS/MS and UPLC-MS/MS techniques were also compared. The method was validated according to the Directive 2002/657/EC and subsequently applied to 56 samples of raw cow milk supplied by the Laboratori Interprofessional Lleter de Catalunya (ALLIC) (Laboratori Interprofessional Lleter de Catalunya, Control Laboratory Interprofessional of Milk of Catalunya).     

Determination of prednisolone in human adipose tissue incubation medium using LC-MS/MS to support the measurement of 11beta-hydroxysteroid dehydrogenase activity

A method for the determination of prednisolone in human adipose tissue incubation medium has been developed, validated and used to support studies designed to measure the activity of 11beta-hydroxysteroid dehydrogenase in human adipose tissue. After incubation, samples (80 microL) were extracted using Oasis HLB microElute SPE plates and the resulting extracts were analyzed using reversed-phase chromatography coupled to an Applied Biosystems Sciex PE API-4000 mass spectrometer with a TurboIonSpray interface (400 degrees C). The method was validated over the calibration range of 0.5-100 ng/mL. Intraday precision and accuracy were 6.1% R.S.D. or less and within 6.3%, respectively. Interday precision and accuracy were 4.2% R.S.D. or less and within 3.6%, respectively. Extraction recovery of prednisolone was greater than 84% over the range of low to high quality control sample concentrations. The validated assay was used to support studies designed to estimate ex vivo 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme activity in human adipose tissue.     

定量比格犬血浆中去甲文拉法辛LC-MS/MS方法的建立及比格犬血浆中富马酸去甲文拉法辛药代动力学研究

目的:建立一种快速灵敏的液质联用方法定量比格犬血浆中去甲文拉法辛,并进行比格犬体内富马酸去甲文拉法辛药代动力学研究,同时与琥珀酸去甲文拉法辛比格犬体内药代参数进行对比。方法:样品处理采用叔丁基甲醚进行液液萃取,液相分离采用Agilent Eclipse Plus C18色谱柱(2.1 mm×100 mm,3.5μm),以乙腈-乙酸铵缓冲液(1 mmol/L)(体积比80∶20)进行等度洗脱。采用单次给药和多次给药试验进行比格犬体内药代动力学研究。结果:此方法具有良好的线性、精密度、准确度,定量下限(0.5 ng/mL)高于文献报道。液液萃取的样品提取方法简便,液质联用分析时间相对较短(3 min)。结论:此方法可成功地应用于比格犬血浆中去甲文拉法辛定量分析。比格犬体内富马酸去甲文拉法辛的药代参数(单次给药和多次给药试验)与对照品琥珀酸去甲文拉法辛没有明显的统计学差异。     

A rapid and sensitive LC-MS/MS assay for the quantitation of deacetyl mycoepoxydiene in rat plasma with application to preclinical pharmacokinetics studies

The purpose of this study was to develop and validate a high-performance liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for analysis of the deacetyl mycoepoxydiene in rat plasma. The analyte and internal standard (I.S.), benorilate, were extracted from rat plasma by precipitation protein and separated on a C(18) column using acetonitrile-0.5% formic acid as mobile phase. Detection was performed using a turbo-spray ionization source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) at an ion voltage of +4800 V. The assay was linear over the concentration range 5-5000 ng/mL with the lowest limit of quantification (LLOQ) of 5 ng/mL. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD<5.8%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to a preclinical pharmacokinetic study in rats after a single intravenous administration of deacetyl mycoepoxydiene 10 mg/kg.     

Clinical validation of an immunoaffinity LC-MS/MS assay for the quantification of a collagen type II neoepitope peptide: A biomarker of matrix metalloproteinase activity and osteoarthritis in human urine

The degradation of type II collagen has been associated with the pathology of osteoarthritis (OA). Matrix metalloproteinases (MMPs) are enzymes that are responsible for catalyzing the degradation of collagen and, therefore, are pursued as potential targets for the treatment of OA. Collagen-derived peptides identified as a reflection of in vivo MMP activity have been investigated as target biomarkers of MMP activity as well as potential biomarkers of OA disease state and/or progression. An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay developed for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues resulting from MMP-13-catalyzed degradation, was validated for clinical use. Validation experiments were designed with attention to specific challenges related to quantification of endogenous analytes. The validated method is sensitive, selective, accurate (<15% relative error) and precise (<15% coefficient of variation) over a linear range of 0.156-7.50 ng/ml. Sample stability and inter- and intrasubject variability were evaluated in the urine of normal and OA populations. The method was applied to analyze human urine samples from clinical studies investigating the utility of uTIINE as a potential biomarker for OA.     

First LC-MS/MS electrospray ionization validated method for the quantification of perindopril and its metabolite perindoprilat in human plasma and its application to bioequivalence study

A high throughput bioanalytical method based on solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been developed for the estimation of perindopril and its metabolite perindoprilat, an angiotensin-converting enzyme inhibitor in human plasma. Ramipril was used as internal standard (IS). The extraction of perindopril, perindoprilat and ramipril from the plasma involved treatment with phosphoric acid followed by solid phase extraction (SPE) using hydrophilic lipophilic balance HLB cartridge. The SPE eluate without drying were analyzed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode to quantify perindopril and perindoprilat in human plasma. The total chromatographic run time was 1.5 min with retention time for perindopril, perindoprilat and ramipril at 0.33, 0.35 and 0.30 min. The developed method was validated in human plasma matrix, with a sensitivity of 0.5 ng/ml (CV, 7.67%) for perindopril and 0.3 ng/ml (CV, 4.94%) for perindoprilat. This method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect especially because the pattern of elution of all the analytes appears as flow injection elution. Sample preparation by this method yielded extremely clean extracts with very good and consistent mean recoveries; 78.29% for perindopril, 76.32% for perindoprilat and 77.72% for IS. The response of the LC-MS/MS method for perindopril and perindoprilat was linear over the range 0.5-350.0 ng/ml for perindopril and 0.3-40 ng/ml for perindoprilat with correlation coefficient, r>/=0.9998 and 0.9996, respectively. The method was successfully applied for bioequivalence studies in human subjects samples with 4 mg immediate release (IR) formulations.     

More than one way to control hair growth: regulatory mechanisms in enterobacteria that affect fimbriae assembled by the chaperone/usher pathway

Many gram-negative enterobacteria produce surface-associated fimbriae that facilitate attachment and adherence to eucaryotic cells and tissues. These organelles are believed to play an important role during infection by enabling bacteria to colonize specific niches within their hosts. One class of these fimbriae is assembled using a periplasmic chaperone and membrane-associated scaffolding protein that has been referred to as an usher because of its function in fimbrial biogenesis. The presence of multiple types of fimbriae assembled by the chaperone/usher pathway can be found both within a single bacterial species and also among different genera. One way of controlling fimbrial assembly in these bacteria is at the genetic level by positively or negatively regulating fimbrial gene expression. This minireview considers the mechanisms that have been described to control fimbrial gene expression and uses specific examples to demonstrate both unique and shared properties of such regulatory mechanisms.